Modulating fatty acid metabolism and composition of CHO cells by feeding high levels of fatty acids complexed using methyl-β-cyclodextrin

Abstract

Chinese Hamster Ovary (CHO) cells are widely used in the pharmaceutical industry to produce therapeutic proteins. Increasing the productivity of CHO cells through media development and genetic engineering is a significant industry objective. Past research demonstrated the benefits of modulating fatty acid composition of CHO cells through genetic engineering. In this study, we describe an alternative approach to modulate fatty acid composition by directly feeding high levels of fatty acids in CHO cell culture. To accomplish this, we developed and optimized a pharmaceutically relevant feeding strategy using methyl-β-cyclodextrin (MBCD) to solubilize fatty acids. To quantify fatty acid composition of CHO cells, a new GC-MS protocol was developed and validated. In fed batch cultures, we found that the degree of saturation of fatty acids in CHO cell mass, i.e. the relative abundances of saturated, monounsaturated and polyunsaturated fatty acids, can be controlled by the choice of fatty acid supplement and feeding strategy. Feeding unsaturated fatty acids such as palmitoleic acid, oleic acid, and linoleic acid had the greatest impact the fatty acid composition of CHO cells, increasing their respective abundances in cell mass by upwards of 25x, 1.5x, and 50x, respectively. ¹³C-Tracing further revealed that the supplemented fatty acids were involved in a range of elongation, desaturation, and β-oxidation reactions to yield both common and uncommon fatty acids such as vaccenic acid and hypogeic acid. Finally, we show that CHO-K1 and CHO-GS cells take up fatty acids solubilized with MBCD at rates comparable to delivery using bovine serum albumin. Taken together, this work paves the way for new feed media formulations containing fatty acids to optimize CHO cell physiology in industrial cell cultures.